The invention relates to improvements in a multiple channel spectrometer capable of quickly analyzing large volumes of samples by the fluorescence emitted by the samples. While many different systems for causing fluorescence emission and processing of the fluorescence may be used, one currently preferred method is known as fluorescence correlation spectroscopy (also known as fluorescence fluctuation spectroscopy). Examples of this technique are as described in Schrof et al. U.S. Pat. No. 5,815,262. Other examples of fluorescence cross-correlation spectroscopy are shown in the articles by Andre Koltermann et al. from the Proceedings of the National Academy of Science, Volume 95, pages 1421-1426, February 1998 entitled xe2x80x9cRapid Assay Processing by Integration of Dual Color Fluorescence Cross-Correlation Spectroscopy: High Throughput Screening for Enzyme Activityxe2x80x9d; and the article by Petra Schwille et al. from the Biophysical Journal, Volume 72, pages 1878-1886, April 1997, entitled xe2x80x9cDual Color Fluorescence Cross-Correlation spectroscopy for Multi Component Diffusional Analysis Solutionxe2x80x9d.
In the Schrof et al. U.S. Pat. No. 5,815,262, fluorescence correlation spectroscopy (FCS) is described in which an excitation laser beam passes through a series of wells (sample chambers) arranged in linear array. The beam is refocused prior to entering each sample chamber, so that fluorescence takes place in a very small area, which area is monitored and sensed to obtain fluorescence data simultaneously from a plurality of samples.
With such a technique, difficulties have been found in the refocusing of light after the laser beam has passed through the first well or sample chamber. Also, possible change of the laser light beam may take place as it passes through the various, separate samples, which may effect the data obtained from particularly the xe2x80x9cdownstreamxe2x80x9d samples in the linear array. Also, two photon excitation, as is used in the above cited patent, generally requires a very expensive laser, while one photon excitation permits the use of cheaper lasers.
Additionally most of the prior art systems utilize a conventional hardware correlator to receive the raw data from multiwell plates in fluorescence correlation spectroscopy. Data from the hardware correlator is passed to the computer processing unit for determination of the parameters of interest, which may include diffusion coefficients and the concentrations of the components. However, several limitations hinder the use of a hardware correlator in this manner. Specifically, when a particulate or an aggregate is present in the solution being analyzed, and it passes through the tiny region illuminated by the focused laser beam, the entire calculated autocorrelation function resulting from the data has to be rejected, as it is altered by the temporary presence of the particulate or aggregate. As the result, the measurement on that specific well from where the data comes has to be rejected, and, more likely, measurement of the entire multiwell plate has to be acquired again.
Also, the raw data may contain useful information and features that are completely and definitively lost once the data has been processed through the hardware correlator. By system of this invention, the user can examine the raw data after they have been acquired. Specifically, it may turn out that higher order correlation functions may be of more interest in describing the molecular interactions occurring in the sample solutions being analyzed through the spectrometer. Thus, if one can keep the raw data, this permits the user to further analyze it with more complicated analysis models when that is desired.
The analysis can be easily implemented in an automated fashion by properly designed software for a spectrometer, particularly for high throughput screening instruments.
Also, the first order autocorrelation function of fluorescence correlation spectroscopy is typically determined by using the time-mode, which is the traditional way of calculating the function. See particularly Thompson, Fluorescence Correlation Spectroscopy, in xe2x80x9cTopics in Fluorescence Spectroscopyxe2x80x9d, Volume 1 (J. R. Lakowicz, Editor), Plenum Press, New York 1991, pages 337-410. Time mode operations tend to limit the precision in determining concentrations, and increase the data acquisition time.
It is desired to perform drug and other screening at very large rates of analysis. For example, current techniques can allow screening up to 50,000 to 100,000 compounds a day. However, it should be desirable in the field of high throughput screening to significantly increase the capacity of spectrometers to process large numbers of compounds. Many of the current high throughput screening apparatus are manufactured by L. J. L. Biosystems of Sunnyvale, Calif.; Aurora Biosciences of San Diego, Calif.; Molecular Devices of Sunnyvale, Calif.; and Packard Instruments of Meriden, Conn.
Fluorescence emission is usually preferred for such high throughput uses due to the overall sensitivity when compared to other techniques such as absorption measurements. Another advantage of using fluorescence is the detection method of availability of a range of fluorophores that can be used as extrinsic probes. Typically, five parameters can be measured when using a fluorescence technique, namely the intensity of the excitation spectra, the intensity of the emission spectra (each at selected wavelengths), the polarization of the excitation spectrum, the quantum yield of fluorescence, and the decay time of the excited level.
Fluorescence correlation spectroscopy was originally proposed by Magde et al., Thermodynamic Fluctuations in a Reacting System: Measurements by Fluorescence Correlation Spectroscopy, Physical Review Letters, Volume 29 (1972), pages 705-708. In this technique, the temporal fluctuations of the detected fluorescence signal (that is time-dependent, spontaneous intensity fluctuations of the fluorescence signal in the typically tiny observation volume) are detected and analyzed to obtain information about the processes occurring on a molecular scale. These intensity fluctuations and the volume under observation may arise from Brownian motion, flow, and chemical reactions. During the past years, fluorescence correlation spectroscopy has been utilized to measure transitional diffusion coefficients, rotational diffusion coefficients, kinetic rate constants, molecular aggregation, and molecular weights. An article by Thompson et al., presents a review of the technique (N. L. Thompson et al., Fluorescence Correlation Spectroscopy, in xe2x80x9cTopics in Fluorescence Spectroscopyxe2x80x9d, Volume 1 (J. R. Lakowicz, Editor Pleanum Press, New York 1991, pages 337-410).
By this invention, an apparatus and method are provided for carrying out high throughput screening of active compounds, typically using fluorescence correlation spectroscopy, although other techniques may be utilized making use of this invention. A fluorescence probe is excited through a one photon or multi photon excitation process. The light source may be lamp such as an xenon arc or deuterium lamp, or a laser such as continuous wave lasers: i.e., argon-ion, krypton-ion, helium-neon, helium-cadmium, or other lasers. Pulsed lasers may also be used such as nitrogen lasers or mode-locked lasers, diode lasers, or lasers placed in an array. In each of the possible radiation sources, the light source should be capable of delivering radiation at a particular wavelength or wavelengths that excite the fluorescence probe through one photon or multi photon excitation processes. Typically, such excitation wavelengths may range from 200 nm. to 5,000 nm.
By this invention, a fluorescence spectrometer is provided which comprises a laser; and at least one beam splitter (which may be a prism-type beam splitter, a fiber optic system, or similar device for accomplishing the beam splitting function) positioned to receive a light beam from the laser and to divide the beam into a plurality of separate, first light portions. Thus, multiple first light beam portions are provided, typically spaced from and extending parallel to each other.
Dichroic mirrors are positioned to separately receive the first light beam portions and to reflect the beam portions at an angle to the first light beam portions, typically perpendicular. A plurality of transparent wall chambers (such as wells) are provided for holding samples to be analyzed. Typically, a conventional multiple well sample plate may be used. Objective lens systems are provided (typically for confocal use), for example microscopes, which systems are respectively positioned in the path of each of the reflected first beam portions, to respectively focus each reflected beam portion to a point within one of the separate, transparent chambers, to elicit a fluorescent response from the sample for testing in the chamber.
Lenses are respectively positioned to receive the fluorescence from the sample for testing (which samples are within the transparent chambers), and to respectively focus the fluorescence at pinholes in respective, opaque partitions. The lenses are positioned to receive the fluorescence which passes back through the objective lens system and the dichroic mirror, and then to focus the fluorescence at a respective pinhole. The dichroic mirror is selected so that it is transparent to at least one wavelength of the fluorescence.
Light detectors are each respectively positioned adjacent to one of the pinholes, with that pinhole being respectively positioned between the lens and the light detector of the particular system, to permit each light detector to sense fluorescence through the pinhole. Electronics are then provided to receive and process signals from each light detector.
The spectrometer of this invention will comprise a plurality of such individual systems, typically four or eight, for simultaneous processing of sample in separate chambers or wells, to contribute to a high throughput system.
Typically, a plate comprises the transparent chambers described above, plus a plurality of other transparent chambers for holding samples, typically carried by the same plate. An x-y movement device carries the plate and permits a first group of the chambers to be respectively and simultaneously exposed to the first light beam portions for analysis of the resulting fluorescence. Then, the x-y device may be moved in a two dimensional, planar manner for exposure of another group of chambers of the plate to the first light beam portions, for their analysis.
Preferably, the system is set up so that fluorescence from each transparent chamber which passes through the objective lens system and the dichroic mirror extends in a straight line, passing further through the objective lens system and the pinhole to the light detector in the same straight line.
Accordingly, all array of such detector systems, comprising the above items to control and process multiple first beam portions, can be used to systematically and simultaneously analyze the contents of multiple chambers of a plate, followed by translation of an x-y table which carries the plate, for analysis of another set of the chambers, until the multiple chamber plate (up to 384 or more such chambers) has been completely and rapidly analyzed. By way of advantage, each of the first beam portions produced by this invention pass through only one chamber and sample per individual analysis, so there is no possibility of chamber data cross contamination resulting from a light beam that passes through multiple chambers to elicit a fluorescent response, as in the patent cited above.
Preferably, the light excitation and detection scheme used in this invention follows the confocal design of microscopy. Data acquisition can be achieved by measuring the time intervals between the photons reaching the detector and building a histogram of the detected counts. The volume in the sample from which photons are detected can be of extremely small volume, for example, about 0.1 to 10 femtoliters, which is on the order of the volume of a bacterium. The photon counting histogram provides the concentrations of the molecular species present in the solution and the number of photons emitted by each species. The invention can also derive simultaneously the autocorrelation function or higher-order correlation functions of the parameters of interest for rapid high throughput. Screening can be acquired on such small observation volumes over a time of about one to five seconds. Direct measurement of single molecules and the kinetics involved over any desired time scale can be achieved by this technique. The lower limit of detection is due to impurities and buffer contamination, plus the effect of the particulate resulting from insoluble compounds.
Thus one can perform fluorescence correlation spectroscopy by acquiring a fluorescence measurement from a sample over time to obtain a histogram of photon counts; deriving the molecular brightness of at lest one molecular species in the sample; and determining he concentration of the species from the data obtained.
Data acquisition and analysis of the data may comprise the steps of: acquiring a fluorescence measurement over time from a sample in the form of electronic raw data. This electronic raw data may be picked up by a light detector, sent to a preamplifier discriminator, and then sent to a computer, where the raw data may be stored, in accordance with this invention, and contrary to the procedure used with the conventional hardware correlator.
The electronically stored raw data is then processed using a first algorithm, without erasing the electronically stored, original raw data. Then, as an advantage of and by means of this invention, the stored raw data may be reprocessed using one or more additional algorithms, or the data may be reprocessed with the first algorithm, without the need of acquiring a complete set of new data as may be required in the prior art.
In fluorescence correlation spectroscopy (FCS), the fluorescence signal F(t) as a function of time is measured as the raw data. The temporal autocorrelation of the fluorescence fluctuations, which is a measurement of the average temporal duration of the fluorescence fluctuations, is determined. Typically, the normalized autocorrelation function is defined as:                               G          ⁡                      (            τ            )                          =                              ⟨                          δ              ⁢                              xe2x80x83                            ⁢                              F                ⁡                                  (                                      t                    +                    τ                                    )                                            ⁢              δ              ⁢                              xe2x80x83                            ⁢                              F                ⁡                                  (                  t                  )                                                      ⟩                                              ⟨                              F                ⁡                                  (                  t                  )                                            ⟩                        2                                              (        1        )            
G(xcfx84) decays in time. The rate of the decay and the shape of the Curve contain information about the mechanisms and the rates of the processes that generate the fluorescence fluctuations. The observed fluctuations of the fluorescence signal obey Poisson statistics with the amplitude of the average fluctuation proportional to N1/2, where N represents the number of molecules in the observation volume. Nanomolar concentrations can be detected. Using FCS with two-photon excitation, the observation volume ranges typically from 0.1 to 1.0xc3x9710xe2x88x9215 liters (or femtoliter, abbreviated as xe2x80x9cflxe2x80x9d). This small observation volume allows the direct measurement of single molecules and the kinetics involved over a time scale extending from hundreds of nanoseconds to seconds or hours. The lower limit of detection is due to impurities and buffer contamination and the effect of the particulate generated from insoluble compounds.
It is generally preferred to acquire the data using a photon-mode technique rather than a time mode technique, although either method may be used in accordance with this invention.
In the photon mode technique, as is known per se, the detector records the time delay between one photon and the next photon from the fluorescence arriving to the detector. In this implementation of the data acquisition, the xe2x80x9cclocksxe2x80x9d are the events to be recorded, and the photons are the starts-stops which define each interval.
On the other hand, in time mode techniques, the detector counts the number of photons collected from the sample in a specific time interval. Typically, the instrument uses about 256 different time intervals. The length of a time interval is specified by the user through the software which controls the operation. Thus, the photons are the xe2x80x9ceventsxe2x80x9d to be recorded at each time interval. The xe2x80x9cclocksxe2x80x9d are the arbitrary starting and ending stops defined by the electronics to create each desired time interval.
By way of advantage, the method of this invention may be used in conjunction with a one photon excitation technique, in which the excited level of the molecule is created by one photon contrary to the situation where two or more photons are needed to create the necessary excitation to achieve fluorescence in the sample. One photon excitation techniques can use significantly less expensive lasers.
In this invention, a data acquisition card may be provided in the computer used for processing the data, in which the data acquisition card acquires the raw data from the light detector and arranges for its storage in the computer memory. Then, the computer may calculate the desired autocorrelation function or higher-order correlation functions. This provides the advantage that bad data may be eliminated without losing the entire data set pertinent to the sample well under examination, contrary to the situation of a hardware correlator. The bad data may result from the presence of a solid particle in the tiny volume being examined for fluorescence. Additionally, the raw data may be rerun after acquisition for recalculation based on higher order correlation functions, if that is needed. Such a data acquisition card can also have the capability of acquiring the data in either the time mode or in the photon mode, as may be desired.
Also, as previously discussed, rapid, multiple, simultaneous analysis of sample chambers may take place in accordance with this invention for high volume screening operations.